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EP1360282B1 - Méthode d'inactivation de la TSE - Google Patents

Méthode d'inactivation de la TSE Download PDF

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Publication number
EP1360282B1
EP1360282B1 EP02726996A EP02726996A EP1360282B1 EP 1360282 B1 EP1360282 B1 EP 1360282B1 EP 02726996 A EP02726996 A EP 02726996A EP 02726996 A EP02726996 A EP 02726996A EP 1360282 B1 EP1360282 B1 EP 1360282B1
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Prior art keywords
tse
temperature
enzyme
solution
agent
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EP1360282A2 (fr
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Neil D.H. Health Protection Agency RAVEN
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Public Health England
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Health Protection Agency
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Priority claimed from GB0100420A external-priority patent/GB0100420D0/en
Priority claimed from GB0104696A external-priority patent/GB0104696D0/en
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/22—Phase substances, e.g. smokes, aerosols or sprayed or atomised substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/015—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone
    • A61L9/02—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone using substances evaporated in the air by heating or combustion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38618—Protease or amylase in liquid compositions only
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the invention is in the field of methods and compositions for the sterilisation of materials and apparatus that may have been contaminated with infectious agents and for detection of those agents.
  • the invention relates to methods for the inactivation and detection of transmissible spongiform encephalopathy (TSE) agents and provides compositions for degrading and detecting TSE located on or within infected materials.
  • TSE transmissible spongiform encephalopathy
  • TSEs Transmissible spongiform encephalopathies
  • CJD Creutzfeld-Jacob disease
  • BSE bovine spongiform encephalopathy
  • Scrapie sheep.
  • TSEs are characterised by the conversion of a normal host protein into a pathogenic protein within the brain tissue of an infected animal.
  • the pathogenic form of protein is often referred to as a prion and is highly resistant to physical and chemical degradation.
  • the prion is believed to be the transmissive agent through which the TSE disease is passed on between animals.
  • Sterilisation of instruments and equipment following potential exposure to TSE infected tissue is of primary importance.
  • surgical equipment such as scalpels, forceps and endoscopes should be thoroughly sterilised before use on patients to avoid disease transmission.
  • TSE transmission is typically observed in cases where infected material is transferred between animals or implanted into an animal. As described previously, incomplete or inadequate sterilisation of surgical instruments can lead to such transfer of infected material between patients. Even the most rigorous chemical cleaning and steam sterilisation procedures can fail to remove blood and tissue from surgical instruments, especially in the jaws or joints offorceps and clamps (Laurenson (1999) The Lancet, 20 Nov). Thus, the risk of unintentional TSE transfer can be unnecessarily high.
  • TSE agents or prions
  • TSE agents are known to be highly resistant to denaturation and degradation, more so than would normally be expected for a protein.
  • Taylor J. Hosp. Infect. (1999) 43 supplement, pp S69-76 reviews a number of methods for inactivating prion proteins.
  • US Patent No. 5, 234, 832 describes the use of a proteolytic enzyme for cleaning and disinfecting medical instruments.
  • a first aspect of the invention provides a method for inactivating a TSE agent comprising exposing the TSE agent to a thermostable proteolytic enzyme at a temperature in the range 50-120°, wherein the enzyme is thermally stable and biologically active at a temperature in the range 50-120°.
  • the methods and compositions of the invention are suitable for the inactivation of TSE agent in apparatus and materials infected or suspected as being infected with TSE agent.
  • the methods and compositions of the invention are suitably used in a prophylactic or precautionary mode, where definite knowledge of infection is uncertain.
  • the method of the invention can be easily incorporated into the standard sterilisation protocols used for preparation of surgical apparatus prior to use its use in surgical procedures.
  • the methods and compositions of the present invention are also suitably used for the inactivation of TSE agents in potentially contaminated clinical waste and culled animal material.
  • this waste material is incinerated at 1000°C, which requires specialised facilities and is expensive. It is an advantage of the present invention that a TSE inactivation procedure can occur at temperatures and in conditions which do not require highly specialised facilities and that the prospects of complete inactivation of the TSE agent are comparable to the more energy intensive and expensive incineration procedures.
  • TSE transmissible spongiform encephalopathy
  • CJD human diseases Creutzfeld-Jacob disease
  • vCJD variant Creutzfeld-Jacob disease
  • Kuru fatal familial insomnia
  • Gerstmann-Straussler-Scheinker syndrome Non-human TSEs include bovine spongiform encephalopathy (BSE), scraple, feline spongiform encephalopathy, chronic wasting disease, and transmissible mink encephalopathy.
  • BSE bovine spongiform encephalopathy
  • vCJD is currently understood to be a human form of BSE, it is apparent that certain TSE agents are capable of crossing the species barrier and that novel TSEs from non-bovine sources could become evident in future.
  • the proteolytic enzyme of the invention is typically a protease but can be suitably any biological polymeric molecule capable of catalysing cleavage of a polypeptide chain.
  • the proteolytic enzyme is a thermostable enzyme, that is, that it demonstrates optimal biological activity at temperatures in excess of the normal mammalian body temperature of 37°C.
  • the enzyme is thermally stable and biologically active, and inactivation is carried out, at temperatures in the range of 50°C to 120°C; and more preferably where the temperature is between 55°C and 85°C. In a specific embodiment of the invention the temperature is about 60°C. In a further specific embodiment the temperature is about 80°C.
  • thermophilic bacteria and archaea suitable for use in the methods and compositions of the invention are obtainable from a number of sources such as thermophilic bacteria and archaea.
  • thermostable proteolytic enzyme is isolated from thermophilic bacteria, hyperthermophilic bacteria and archaea.
  • Suitable organisms for extraction of proteolytic enzymes for use in the invention include Thermotoga maritima; Thermotoga neopolitana; Thermotoga thermarum; Fervidobacterium islandicum; Fervidobacterium nodosum; Fervidobacterium pennivorans; Thermosipho africanus; Aeropyrum pemix; Thermus flavus; pyrococcus spp.; Sulfolobus solfataricus; Desulfurococcus; Bacillus thermoproteolyticus; Bacillus stearo-thermophilus; Bacillus sp. 11231; Bacillus sp. 11276; Bacillus sp. 11652; Bacillus sp. 12031; Thennus aquaticus; Thermus caldophilus; Thermus sp. 16132; Thermus sp. 15673; and Thermus sp. Rt41A .
  • thermostable proteases are not the only sources of thermostable proteases. Indeed, some organisms that are not considered to be truly thermophilic can also express thermostable proteolytic enzymes. Such organisms are commonly termed thermodurable in that although they do not choose to live in conditions of high temperature, they can withstand high temperatures for limited periods. A number of Bacillus species fall in the category of thermodurability and are known to produce thermostable subtilisin-type proteases.
  • the pH at which the inactivation is performed can range from acid to alkaline, but is typically in the region of pH 8-13, preferably pH greater than 9 and more preferably around pH 12.
  • the thermostable protease is active in a pH range from acid to alkaline, but typically is optimally active in the region of pH 8-13, and preferably pH greater than 9 and more preferably around pH 12.
  • the proteolytic enzyme is extracted from a culture of the thermophilic bacteria or archaea.
  • the culture is suitably maintained under the optimal conditions for the organism typically within a bioreactor.
  • a continuous source of the organism can be maintained, allowing proteolytic enzyme to be obtained whenever needed.
  • the gene encoding a thermostable proteolytic enzyme is isolated from the source organism. Bacillus thermoproteolyticus .
  • the gene is used to generate a recombinant expression construct, typically a plasmid, which is transformed into a host organism, Escherichia coli .
  • the transformed E. coli is grown in a bioreactor and when at an appropriate cell density the expression of the plasmid construct is initiated and the proteolytic enzyme harvested using standard methods.
  • the recombinant expression route allows for the production of the proteolytic enzyme product under less extreme conditions of temperature than would be required for the original source organism.
  • thermostable protease genes in order to increase thermal stability or biological activity or for some other purpose.
  • activity of a thermostable proteolytic enzyme can be readily optimised for use in the methods and compositions of the invention.
  • a second aspect of the invention uses the method for sterilising apparatus, comprising the step of exposing the apparatus to a solution comprising a thermostable proteolytic enzyme.
  • the term "sterilising” is commonly understood to mean the procedure by which living organisms are removed from or killed in a substrate, such as a piece of equipment or a solution.
  • the TSE agent, or prion is not technically . considered to be a living organism, in the sense that a bacterium or virus is, because it does not apparently contain any genetic material.
  • the transmission of the TSE pathogenic agent between animats does result in disease.
  • the term "sterilising” as used herein is applied to the procedure by which both pathogenic agents (such as TSE agents) and living organisms are rendered non-infective or removed from or killed in a substrate.
  • the method of the invention comprises maintaining the sterilising solution at a temperature below 100°C, preferably at least 45°C and more preferably between 45°C and 85°C.
  • the pH of the sterilising solution can range from acid to alkaline, but is typically in the region of pH 8-13, at least pH 9 and more preferably around pH 12.
  • the thermostable protease is active in a pH range from acid to alkaline, but typically is optimally active in the region of pH 8-13, at least pH9 and more preferably around pH 12.
  • the sterilising solution is applied to the apparatus as a spray.
  • the advantage of this mode of application is that, larger surface areas of apparatus, operating tables or even walls of rooms (for example in abattoirs) can be treated with the sterilising solution of the invention.
  • the solution will be heated to an optimal temperature, for example between 60°C to 80°C, before being sprayed onto the surface that is to be sterilised, that surface being optionally heated in advance.
  • the apparatus is immersed in the sterilising solution for a predetermined period of time.
  • the temperature of the solution is typically optimised prior to immersion of the contaminated apparatus. It is optional to include ultrasonication means in the immersion bath to enable ultrasonic cleaning to occur at the same time as treatment with the sterilising solution of the invention.
  • the invention provides for a method of sterilising material, comprising exposing said material to a first solution comprising a thermostable proteolytic enzyme; and then exposing the apparatus to at least a second solution comprising a second thermostable proteolytic enzyme.
  • the material is typically apparatus, surgical or meat rendering equipment. or TSE infected biological waste.
  • the conditions in each step can be optimised to ensure maximum inactivation of any TSE agent present.
  • the temperatures and/or pH of successive steps can be different.
  • the proteolytic enzymes in the first and second (and optionally more) solutions are the same, or are different.
  • the invention uses a composition for inactivating a TSE agent, comprising a thermostable proteolytic enzyme.
  • the composition of the invention further comprises a buffering agent.
  • the buffering agent has a pK a of between 8 and 13.
  • Alkaline buffers suitable for use in the method of the invention include 4-cyclohexylamino-1-butanesulfonic acid (CABS) which has a pKa of 10.7 at 25°C, and 3-cyclohexylamino-1-propanesulfonic acid (CAPS) which has a pKa of 10.4 at 25°C.
  • CABS 4-cyclohexylamino-1-butanesulfonic acid
  • CAS 3-cyclohexylamino-1-propanesulfonic acid
  • the composition comprises sufficient sodium hydroxide or other alkaline agent to adjust the pH of the composition to alkaline, preferably to at least pH 9 and more preferably around pH 12.
  • An advantage of the invention is its use in degrading TSE and similar agents, and it has been found in operation of particular embodiments of the invention that TSE-contaminated material has been successfully decontaminated using a combination of elevated temperature of around 50°C to 70°C and alkaline pH of around 9 to 12, with a thermostable, alkophilic proteolytic enzyme. Whilst it is on occasion possible to achieve some decontamination by extremely high temperature alone, it is of significant benefit to be able to inactivate TSE whilst avoiding extreme conditions, such as extremes of temperature, which lead to damage to the equipment being decontaminated.
  • the invention also provides dimer removal using protease degradation which is optionally enhanced by environmental conditions - high temperature, extremes of pH, and/or the use of detergents (e.g. SDS).
  • protease degradation is optionally enhanced by environmental conditions - high temperature, extremes of pH, and/or the use of detergents (e.g. SDS).
  • combinations of proteases and/or other enzymes can be used.
  • better degradation of the infective agent may be achieved by the addition of lipases, peptidases, glycosylases, nucleases and other enzymes.
  • a dimer cross-reactive antibody can be used in conjunction with a suitable detection system, one example being a sensitive in vitro detection system currently available from Invitrogen, referred to as Western Breeze, to confirm removal prior to further mouse bioassays.
  • a suitable detection system one example being a sensitive in vitro detection system currently available from Invitrogen, referred to as Western Breeze, to confirm removal prior to further mouse bioassays.
  • the monomer is apparently completely removed by protease digestion. Under these conditions, however, samples which were subsequently used in vivo did not prevent infection and may even have enhanced its onset. There must, therefore, be another source of infection other than the monomer. Since the monomer can be removed (or at worst dramatically reduced in concentration) this indicates a primary role in infectivity for the dimer - either alone or in combination with the monomer.
  • BSE strain 301V
  • VM mouse strain selected for use in the study was obtained from Dr David Taylor (Institute of Animal Health, Edinburgh). Six pairs were introduced into a dedicated room within an animal facifity. Mice were screened for their health status and a breeding programme initiated.
  • BSE (301V) infectious mouse brain (IAH, Edinburgh) was prepared for inoculation by crude homogenisation followed by passage of the brains through increasingly fine gauge luer-locked needles (from 21G-27G) to and from a contained safety syringe into a closed septum-topped vial. This procedure was carried out in a validated safety cabinet within a containment level 3 (CL3) laboratory immediately prior to intracerebral inoculation of the VM mice.
  • the anaesthetised (alphadolone/alphaxalone) mice were inoculated intracerebrally via 26 gauge needles with 20 ⁇ l of the mouse brain homogenate preparation. Forty-nine out of fifty mice survived this procedure. These were retained to allow incubation of the agent and the generation of the required quantity of high-titre infectious material.
  • Organisms selected for the production of biomass were chosen for the production of biomass in order to provide as broad a selection of thermostable proteolytic enzymes as possible.
  • Thermophiles were also selected to cover a wide range of growth pH, encompassing pH optima from pH2.5 to pH11.5. The majority of organisms were grown in batch culture, however, where the growth requirements of the organism were particularly fastidious, continuous culture was used (Raven and Sharp (1997) Applied Microbial Physiology: A Practical Approach, Ch. 2, Eds. Stanbury and Rhodes, OUP pp.23-52).
  • batch culture volumes of between 20L and 120L were employed to achieve the desired amount of cell paste.
  • the continuous culture system utilised either a 2L or 5L working volume gas lift bioreactor constructed entirety of glass and PTFE. More prolific organisms such as Bacillus spp., and Thermus spp., were pre-screened to select those with high levels of protease activity prior to their culture on a larger scale.
  • a quick and sensitive fluorometric protease assay utilising microtitre plates was. adopted for this purpose to permit high through-put screening (EnzChekTM, Molecular Probes, Leiden, Netherlands).
  • Culture biomass was harvested by continuous centrifugation (Contifuge StratosTM, Kendro Laboratory Products, Bishop Stortford, UK) and stored at -80°C. Culture supernatants were concentrated with a 10KDa cut off tangential flow filter (Pall filtration, Portsmouth, UK). Proteins were precipitated with ammonium sulphate (90% saturation) and stored at -80°C.
  • a rapid protease screening and purification technique was required in order to process all of the crude protein preparations after the biomass production stage.
  • a dye-ligand affinity chromatography system was used for this purpose (PIKSI MTM, Affinity Chromatography Ltd., Isle of Man, UK). Initially, each crude ammonium sulphate precipitate was dissolved in buffer and passed through a desalting column. Each sample was then loaded onto the RIKSI M test kit, which contained 10 different affinity ligands. Fractions were then assayed for protease activity to determine the most suitable matrix for purification of the protease, either by positive binding of the target molecule and then elution, or by negative binding of contaminants.
  • the fluorometric protease assay utilises casein derivatives that are heavily labelled with green fluorescent BODIPY FL in which the conjugates' fluorescence is almost totally quenched. Protease catalysed hydrolysis releases the highly fluorescent label and the resultant fluorescence can be measured on a fluorometric microplate reader (Labsystems Fluoroscan IITM). The increase in fluorescence is proportional to protease activity and was compared with that of a standard protease (Protease X, Sigma-Aldrich, Poole, UK).
  • thermostable proteases were analysed (see Table 1). Direct characterisation of activity was carried out using the closest non-infectious analogue to BSE (301V) - infectious mouse brain homogenate available as substrate, i.e. normal VM mouse brain homogenate. Initial digests of total uninfected mouse brain homogenate (mbh) were performed over thirty minutes at 60°C and at pH7.0. The samples were then boiled under reducing conditions and analysed by SDS-PAGE on pre-cast NuPage 4 - 12% Bis-Tris gels (NovexTM, San Diego, US). Gels were fixed using standard procedures and the proteins visualised using the Novex colloidal blue staining kit.
  • Figs. 1 and 2 show the activity profiles for a sample protease, Bacillus protease M. As can be seen the enzyme is moderately thermophilic and gives complete digestion at temperatures up to 70°C. Similarly the pH profile indicates a preference for neutral or alkaline conditions.
  • Mbh proteins were transferred onto nitrocellulose and blocked overnight in PBS-Tween (PBST) + 3% skimmed milk powder.
  • PBST PBS-Tween
  • the membrane was washed (x3 in PBST) and incubated for 1 hour with 6H4 anti-human recombinant PrP monoclonal antibody (Prionics, Zurich, Switzerland). After a second washing step, anti-mouse HRP-conjugate was added and the membrane incubated for 1 hour. Washing was repeated and the antibody reaction visualised by addition of TMB (Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Press).
  • Figures 5 and 6 show an SDS-PAGE and an immunoblot of undigested mbh respectively. Although there is some non-specific background, dark bands indicating the presence of mouse prion can clearly be seen at the expected molecular weight ( ⁇ 33-35kDa). Distinct bands are also visible at ⁇ 66-70kDa, which may correspond to prion dimers previously reported (Safar et al . (1990) Proc. Natl. Acad. Sci. USA, 87:pp6373-6377).
  • mice Eighty days post challenge onward, mice were subject to daily clinical scoring to detect clinically affected mice as early as possible. A single mouse died of an unknown cause prior to this period. The remainder exhibited the expected disease progression and were sacrificed between 110 and 130 days post challenge. The brains were removed aseptically and stored frozen until required. Forty-eight BSE (301V) infectious VM mouse brains were homogenised in four volumes of PBS within a contained homogeniser then passed sequentially through increasingly fine gauge needles (21G to 26G) until free flowing. A sample with a further 2-fold dilution (1:9 mouse brain: PBS) was prepared for titration of infectivity. Over 800 x 0.1ml aliquots of BSE (301V) infectious mouse brain homogenate were prepared. These procedures were again carried out under rigorous class III containment, inducing the wearing of positive pressure respirators.
  • mice received titration doses of the infectious mouse brain homogenate preparation at 10 fold dilutions from 10 -1 - 10 -8 .
  • a further group of 25 mice were challenged with uninfectious mouse brain homogenate as a control. All mice were inoculated under anaesthetic using 26G x 3/8" (0.95cm) needles with plastic sleeve guards cut off 2mm below bevel in a Class 2 cabinet with the use of an injection guard. The mice were then left to incubate the BSE (301V) agent for extended periods some in excess of a year.
  • the initial titre of the infectious mouse brain homogenate preparation was established retrospectively once all incubations (clinical monitoring at 80 days onwards) were complete.
  • BSE (301 V)-infected mouse brain homogenate was digested at neutral pH and 60°C for 30 minutes with protease. Total protein digests were run on SDS-PAGE and transferred by Western blotting to nitro-cellulose membranes. These were cut into strips and probed with CAMR anti-prion antibodies (produced in rabbits). A second generic antibody (goat anti-rabbit) was conjugated to horseradish peroxidase and used with detection by TMB colorimetric substrate.
  • Blot 1 uses a polyclonal antibody raised against a PPD-conjugated peptide corresponding to an N-terminal region of the prion molecule. None is seen in the lanes. This section of the protein is susceptible to proteolysis, so it is not surprising to see nothing in the lanes (2 & 3) - see figure 9, blot 1 on left hand side. Lane 1 is a molecular weight marker.
  • Blot 2 has a second antibody raised against a peptide sequence further into the prion molecule. This shows at least 9 bands of varying intensity, approximately equidistant, at a molecular, weight corresponding to a prion dimer with a range of glycosylation states - see blot 2 on figure 9.
  • Blot 3 antibody shows similar profile; blot 4 is also shown but its results are too poor quality to draw any conclusions - see blots 3 and 4 on figure 9.
  • Blots 5 and 6 shown on figure 10 with the control blot 7, again show the multi-banded pattern
  • Blot 1 shows molecular weight markers in lanes 1 and 5.
  • Lane 2 is recombinant murine PrP showing recombinant murine PrP oligomers.
  • Lane 3 shows lack of antibody response to protease-digested infectious mouse brain homogenate.
  • Lane 4 is the antibody response in the undigested control.
  • Blot 2 is as above but shows the previous banding pattern in the protease digested sample.
  • Blot 3 shows the antibody 3 response. Here there is some response to recombinant murine PrP (lane 2). Lane 3 shows not only the multiple (dimeric PrP) banding pattern, but also some monomeric PrP response.
  • Blot 7 is the 6H4 mAb antibody control. Here there is good detection of recombinant murine PrP oligomers (lane 2). Lane 3 shows the heavily diglycosylated form of limitedly protease-treated PrP Sc , plus the more minor monoglycosylated and non-glycosylated forms typical of BSE (301V) strain. No 'dimer' detection is apparent.
  • the polyclonal sera were produced by immunisation of rabbits with synthesised prion mimetic peptides. These peptides were designed based on regions of high homology between human, mouse and bovine prion protein amino acid sequences.
  • the sequences producing the dimer-reactive antibodies were as follows:
  • the peptides were synthesised with a cysteine at both ends (see above) and with a cysteine at one end onty. This method was used in order to present both the linear form and a loop structure of the antigen on the surface of the carrier protein.
  • the peptides were synthesised commercially and coupled to the carrier protein PPD (purified protein derivative), derived from an attenuated strain of the bacterium Mycobacterium bovis , which is lyophilised and used to conjugate to the peptide via a linker.
  • PPD purified protein derivative
  • Anti-prion polyclonal antibodies were produced as follows:
  • the rabbits were injected with reconstituted freeze-dried Bacillus Calmette-Guerin (BCG) vaccine for intradermal use.
  • BCG Bacillus Calmette-Guerin
  • a dose of 0.1ml of reconstituted BCG vaccine was given in two sites in the scruff of the neck of the rabbit.
  • each peptide-PPD conjugate was measured (0.3mg of each of the 1 cysteine and 2 cysteine versions) and dissolved in 1ml of sterile 0.9% saline.
  • a boost injection was given comprising of the peptide-PPD conjugates prepared as in step 3 and 4.
  • the boost injections consist of four 0.25ml injections into the scruff of the neck of each rabbit.
  • a second boost injection was given 4-6 weeks after the first.
  • a 4ml test bleed was taken 7-14 days after the fourth boost injection and antibody titre determined by ELISA.
  • Terminal exsanguination was carried out and blood collected.
  • the serum was separated by centrifugation and stored at -20°C.
  • the immunoassay plate was coated with the same peptides conjugated to a different carrier protein (KLH) in order to differentiate the response to the peptide from the response to the carrier protein.
  • KLH carrier protein
  • Analagous steps may also be used to prepare a monoclonal antibody. This could be achieved using a method such as described in Antibodies - A Laboratory Manual, Ed Harlow and David Lane, 1988 (Cold Spring Harbor Laboratory).

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Claims (33)

  1. Procédé pour inactiver un agent de l'encéphalopathie spongiforme transmissible (TSE) comprenant l'exposition de l'agent de la TSE à une enzyme protéolytique thermostable à une température dans la plage de 50 à 120°C, dans lequel l'enzyme est thermiquement stable et biologiquement active à une température dans la plage de 50 à 120°C.
  2. Procédé selon la revendication 1, dans lequel l'enzyme est thermiquement stable et biologiquement active à une température entre 55°C et 85°C.
  3. Procédé selon la revendication 2, dans lequel l'enzyme est thermiquement stable et biologiquement active à une température d'environ 60°C.
  4. Procédé selon la revendication 2, dans lequel l'enzyme est thermiquement stable et biologiquement active à une température d'environ 80°C.
  5. Procédé selon l'une quelconque des revendications 1 à 4, comprenant l'exposition de l'agent de la TSE à l'enzyme à une température entre 55°C et 85°C.
  6. Procédé selon la revendication 5, comprenant l'exposition de l'agent de la TSE à l'enzyme à une température d'environ 60°C.
  7. Procédé selon la revendication 5, comprenant l'exposition de l'agent de la TSE à l'enzyme à une température d'environ 80°C.
  8. Procédé selon l'une quelconque des revendications 1 à 7, comprenant l'exposition de l'agent de la TSE à l'enzyme à un pH alcalin.
  9. Procédé selon la revendication 8, dans lequel le pH est entre 8 et 13.
  10. Procédé selon la revendication 8, dans lequel le pH est supérieur à 9.
  11. Procédé selon la revendication 8, dans lequel le pH est environ 12.
  12. Procédé selon l'une quelconque des revendications précédentes, dans lequel l'agent de la TSE est un prion.
  13. Procédé selon l'une quelconque des revendications précédentes, dans lequel la TSE est choisie dans le groupe consistant en la maladie de Creutzfeld-Jacob ; la variante de la maladie de Creuzfeld-Jacob ; le kuru ; l'insomnie fatale familiale ; le syndrome de Gerstmann-Straussler-Scheinker ; l'encéphalopathie spongiforme bovine ; la tremblante du mouton ; l'encéphalopathie spongiforme féline ; la maladie du dépérissement chronique; et l'encéphalopathie transmissible du vison.
  14. Procédé selon l'une quelconque des revendications précédentes, dans lequel l'enzyme protéolytique est obtenue à partir d'un organisme thermophile.
  15. Procédé selon la revendication 14, dans lequel l'organisme thermophile est choisi dans le groupe consistant en les archéobactéries, les bactéries hyperthermophiles et les bactéries thermophiles.
  16. Procédé selon la revendication 14, dans lequel l'organisme thermophile est choisi dans le groupe consistant en Thermotoga maritima ; Thermotoga neopolitana ; Fervidobacterium islandicum ; Fervidobacterium nodosum ; Fervidobacterium pennivorans ; Thermosipho africanus ; Aeropyrum pernix ; Thermus flavus ; Pyrococcus spp ; Sulfolobus solfataricus ; Desulfurococcus ; Bacillus thermoproteolyticus ; Bacillus stearo-thermophilus ; Bacillus sp. 11231 ; Bacillus sp. 11276 ; Bacillus sp. 11652 ; Bacillus sp. 12031 ; Thermus aquaticus ; Thermus caldophilus ; Thermus sp. 16132 ; Thermus sp. 15673 ; et Thermus sp. Rt41A.
  17. Procédé selon l'une quelconque des revendications précédentes pour l'inactivation de la TSE dans les déchets cliniques.
  18. Procédé selon l'une quelconque des revendications précédentes pour l'inactivation de la TSE dans les matières animales éliminées.
  19. Procédé selon l'une quelconque des revendications précédentes, comprenant l'exposition de l'agent de la TSE à l'enzyme à une température dans la plage de 50 à 120°C, et dans lequel le pH est supérieur à 9.
  20. Procédé de stérilisation d'un appareil, comprenant un procédé selon l'une quelconque des revendications 1 à 19.
  21. Procédé selon l'une quelconque des revendications 1 à 20, dans lequel l'enzyme protéolytique thermostable est en solution.
  22. Procédé selon la revendication 21, dans lequel la température de la solution est maintenue en dessous de 100°C.
  23. Procédé selon la revendication 21 ou 22, dans lequel la solution est appliquée comme une vaporisation.
  24. Procédé selon la revendication 21 ou 22, dans lequel l'appareil contaminé est immergé dans la solution.
  25. Procédé selon l'une quelconque des revendications 1 à 24, dans lequel l'agent de la TSE est exposé à une première solution comprenant une enzyme protéolytique qui est thermiquement stable et biologiquement active dans la plage de 50 à 120°C ; et est ensuite exposé à une seconde solution comprenant une enzyme protéolytique qui est thermiquement stable et biologiquement active dans la plage de 50 à 120°C.
  26. Procédé selon la revendication 25, dans lequel la première et la seconde enzymes protéolytiques sont la même.
  27. Procédé selon la revendication 25, dans lequel la première enzyme protéolytique est différente de la seconde enzyme protéolytique.
  28. Procédé selon l'une quelconque des revendications 25 à 27, dans lequel le pH de la première solution est différent du pH de la seconde solution.
  29. Procédé selon l'une quelconque des revendications 25 à 28, dans lequel la température de la première solution est différente de la température de la seconde solution.
  30. Procédé selon l'une quelconque des revendications 1 à 29, comprenant en outre la confirmation de l'inactivation de la TSE en confirmant la suppression du dimère du prion.
  31. Procédé selon la revendication 30, comprenant le sondage avec un anticorps spécifique au dimère du prion.
  32. Utilisation d'une composition comprenant une enzyme protéolytique qui est thermiquement stable et biologiquement active dans la plage de 50 à 120°C pour stériliser du matériel contaminé par un agent de la TSE.
  33. Utilisation d'une enzyme protéolytique qui est thermiquement stable et biologiquement active dans la plage de 50 à 120°C dans la fabrication d'une composition pour inactiver un agent de la TSE.
EP02726996A 2001-01-08 2002-01-08 Méthode d'inactivation de la TSE Revoked EP1360282B1 (fr)

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DK02726996T DK1360282T3 (da) 2001-01-08 2002-01-08 Fremgangsmåde til inaktivering af TSE
EP04025721A EP1577382A3 (fr) 2001-01-08 2002-01-08 Détection de l'infectiosité de TSE

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GB0100420 2001-01-08
GB0100420A GB0100420D0 (en) 2001-01-08 2001-01-08 Degradation of TSE and similar agents
GB0104696 2001-02-26
GB0104696A GB0104696D0 (en) 2001-02-26 2001-02-26 Degradation of tse and similar agents
PCT/GB2002/000052 WO2002053723A2 (fr) 2001-01-08 2002-01-08 Degradation et detection de l'infectivite de la tse

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AUPR293801A0 (en) * 2001-02-07 2001-03-01 Novapharm Research (Australia) Pty Ltd Prion disinfection
US7166471B2 (en) 2001-05-31 2007-01-23 Arete Associates Misfolded protein sensor method in body fluids
DE10203225A1 (de) 2002-01-28 2003-07-31 Weigert Chem Fab Reinigung chirurgischer Instrumente
ATE490313T1 (de) 2002-10-24 2010-12-15 Meiji Seika Kaisha Abbauverfahren für ein schwer abbaubares protein
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GB2394663B (en) * 2002-11-01 2006-07-12 Medical Res Council Prion decontamination
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CA2597912C (fr) 2005-02-15 2013-06-04 Adlyfe, Inc. Procede de detection de proteines et de prions a repliement incorrect
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ES2237675T3 (es) 2005-08-01
WO2002053723B1 (fr) 2003-02-13
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